Evolution of reactive oxygen species cellular targets for plant development.

Reactive oxygen species (ROS) are the key players in regulating developmental processes of plants. Plants have evolved a large array of gene families to facilitate the ROS-regulated developmental process in roots and leaves. However, the cellular targets of ROS during plant evolutionary development are still elusive. Here, we found early evolution and large expansions of protein families such as mitogen-activated protein kinases (MAPK) in the evolutionarily important plant lineages. We review the recent advances in interactions among ROS, phytohormones, gasotransmitters, and protein kinases. We propose that these signaling molecules act in concert to maintain cellular ROS homeostasis in developmental processes of root and leaf to ensure the fine-tuning of plant growth for better adaptation to the changing climate.

Global identification of natural antisense transcripts in Gossypium hirsutum and Gossypium barbadense under chilling stress.

Natural antisense transcripts (NATs) in model plants have been recognized as important regulators of gene expression under abiotic stresses. However, the functional roles of NATs in crops under low temperature are still unclear. Here, we identified 815 and 689 NATs from leaves of Gossypium hirsutum and G. barbadense under chilling stress. Among those, 224 NATs were identified as interspecific homologs between the two species. The correlation coefficients for expression of NATs and their cognate sense genes (CSG) were 0.43 and 0.37 in G. hirsutum and G. barbadense, respectively. Furthermore, expression of interspecific NATs and CSGs alike was highly consistent under chilling stress with correlation coefficients of 0.90-0.91. Four cold-associated NATs were selected for functional validation using virus-induced gene silencing (VIGS). Our results suggest that CAN1 engage in the molecular regulation of chilling stress by regulating SnRK2.8 expression. This highly conserved NAT have valuable potential for applications in breeding cold-tolerant cotton.

RVE2, a new regulatory factor in jasmonic acid pathway, orchestrates resistance to Verticillium wilt.

Verticillium dahliae, one of the most destructive fungal pathogens of several crops, challenges the sustainability of cotton productivity worldwide because very few widely-cultivated Upland cotton varieties are resistant to Verticillium wilt (VW). Here, we report that REVEILLE2 (RVE2), the Myb-like transcription factor, confers the novel function in resistance to VW by regulating the jasmonic acid (JA) pathway in cotton. RVE2 expression was essentially required for the activation of JA-mediated disease-resistance response. RVE2 physically interacted with TPL/TPRs and disturbed JAZ proteins to recruit TPL and TPR1 in NINJA-dependent manner, which regulated JA response by relieving inhibited-MYC2 activity. The MYC2 then bound to RVE2 promoter for the activation of its transcription, forming feedback loop. Interestingly, a unique truncated RVE2 widely existing in D-subgenome (GhRVE2D) of natural Upland cotton represses the ability of the MYC2 to activate GhRVE2A promoter but not GausRVE2 or GbRVE2. The result could partially explain why Gossypium barbadense popularly shows higher resistance than Gossypium hirsutum. Furthermore, disturbing the JA-signalling pathway resulted into the loss of RVE2-mediated disease-resistance in various plants (Arabidopsis, tobacco and cotton). RVE2 overexpression significantly enhanced the resistance to VW. Collectively, we conclude that RVE2, a new regulatory factor, plays a pivotal role in fine-tuning JA-signalling, which would improve our understanding the mechanisms underlying the resistance to VW.

A truncated ETHYLENE INSENSITIVE3-like protein, GhLYI, regulates senescence in cotton.

Numerous endogenous and environmental signals regulate the intricate and highly orchestrated process of plant senescence. Ethylene (ET), which accumulates as senescence progresses, is a major promoter of leaf senescence. The master transcription activator ETHYLENE INSENSITIVE3 (EIN3) activates the expression of a wide range of downstream genes during leaf senescence. Here, we found that a unique EIN3-LIKE 1 (EIL1) gene, cotton LINT YIELD INCREASING (GhLYI), encodes a truncated EIN3 protein in upland cotton (Gossypium hirsutum L.) that functions as an ET signal response factor and a positive regulator of senescence. Ectopic expression or overexpression of GhLYI accelerated leaf senescence in both Arabidopsis (Arabidopsis thaliana) and cotton. Cleavage under targets and tagmentation (CUT&Tag) analyses revealed that SENESCENCE-ASSOCIATED GENE 20 (SAG20) was a target of GhLYI. Electrophoretic mobility shift assay (EMSA), yeast 1-hybrid (Y1H), and dual-luciferase transient expression assay confirmed that GhLYI directly bound the promoter of SAG20 to activate its expression. Transcriptome analysis revealed that transcript levels of a series of senescence-related genes, SAG12, NAC-LIKE, ACTIVATED by APETALA 3/PISTILLATA (NAP/ANAC029), and WRKY53, are substantially induced in GhLYI overexpression plants compared with wild-type (WT) plants. Virus-induced gene silencing (VIGS) preliminarily confirmed that knockdown of GhSAG20 delayed leaf senescence. Collectively, our findings provide a regulatory module involving GhLYI-GhSAG20 in controlling senescence in cotton.

Biotinylated Tn5 transposase-mediated CUT&Tag efficiently profiles transcription factor-DNA interactions in plants.

In contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)-DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase-mediated CUT&Tag (B-CUT&Tag) to produce high-quality libraries for interrogating TF-DNA interactions. B-CUT&Tag combines streptavidin-biotin-based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B-CUT&Tag to probe genome-wide DNA targets of Squamosa promoter-binding-like protein 9 (SPL9), a well-established TF in Arabidopsis; the resulting profiles were efficient and consistent in demonstrating its well-established target genes in juvenile-adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth-defence trade-offs. In addition, we established a method for applying qPCR after CUT&Tag (B-CUT&Tag-qPCR) and successfully validated the binding of SPL9 in Arabidopsis and PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF-chromatin interactions that is widely applicable to the annotation of cis-regulatory elements for crop improvement.

A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5.

Strand-specific RNA-seq is a powerful tool for the discovery of novel transcripts, annotation of genomes, and profiling of gene expression levels. Tn5 transposase has been successfully applied in massive-scale sequencing projects; in particular, Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. We developed a novel dU-adaptor-assembled Tn5-mediated strand-specific RNA-sequencing protocol and compared this method with the leading dUTP method in terms of experimental procedure and multiple quality metrics of the generated libraries. The results showed that the dU-Tn5 method is easy to operate and generates a strand-specific RNA-seq library of comparable quality considering library complexity, strand-specificity, evenness, and continuity of annotated transcript coverage. We also evaluated the performance of the dU-Tn5 method in identifying nitrogen-responsive protein-coding genes and long non-coding RNAs in soybean roots. The results indicated that ~62-70% of differentially expressed genes detected from conventional libraries were also detected in dU-Tn5 libraries, indicating good agreement of our method with the current standard; moreover, their fold-changes were highly correlated (R>0.9). Thus, our method provides a promising 'do-it-yourself' stranded RNA-seq procedure for gene expression profiling.

Genome-Wide Transcriptome Analysis Revealing the Genes Related to Sugar Metabolism in Kernels of Sweet Corn.

Sugar metabolism influences the quality of sweet corn (Zea mays var. saccharate Sturt) kernels, which is a major goal for maize breeding. In this study, the genome-wide transcriptomes from two supersweet corn cultivars (cv. Xuetian 7401 and Zhetian 11) with a nearly two-fold difference in kernel sugar content were carried out to explore the genes related to kernel sugar metabolism. In total, 45,748 differentially expressed genes (DEGs) in kernels and 596 DEGs in leaves were identified. PsbS, photosynthetic system II subunit S, showed two isoforms with different expression levels in leaf tissue between two cultivars, indicating that this gene might influence sugar accumulation in the kernel. On the other hand, hexokinases and beta-glucosidase genes involved in glycolysis, starch and sucrose metabolism were found in developing kernels with a genome-wide transcriptome analysis of developing kernels, which might contribute to the overaccumulation of water-soluble polysaccharides and an increase in the sweetness in the kernels of Xuetian 7401. These results indicated that kernel sugar accumulation in sweet corn might be influenced by both photosynthesis efficiency and the sugar metabolism rate. Our study supplied a new insight for breeding new cultivars with high sugar content and laid the foundation for exploring the regulatory mechanisms of kernel sugar content in corn.

Absence of CG methylation alters the long noncoding transcriptome landscape in multiple species.

The noncoding regions throughout the genome are in large part comprised of transposable elements (TEs), some of which are functionalized with long intergenic noncoding RNAs (lincRNAs). DNA methylation is predominantly associated with TEs, but little is known about its contribution to the transcription of lincRNAs. Here, we examine the lincRNA profiles of DNA methylation-related mutants of five species, Arabidopsis, rice, tomato, maize, and mouse, to elucidate patterns in lincRNA regulation under altered DNA methylation status. Significant activation of lincRNAs was observed in the absence of CG DNA methylation rather than non-CG. Our study establishes a working model of the contribution of DNA methylation to regulation of the dynamic activity of lincRNA transcription.

Neofunctionalization of a polyploidization-activated cotton long intergenic non-coding RNA DAN1 during drought stress regulation.

The genomic shock of whole-genome duplication (WGD) and hybridization introduces great variation into transcriptomes, for both coding and noncoding genes. An altered transcriptome provides a molecular basis for improving adaptation during the evolution of new species. The allotetraploid cotton, together with the putative diploid ancestor species compose a fine model for study the rapid gene neofunctionalization over the genome shock. Here we report on Drought-Associated Non-coding gene 1 (DAN1), a long intergenic noncoding RNA (lincRNA) that arose from the cotton progenitor A-diploid genome after hybridization and WGD events during cotton evolution. DAN1 in allotetraploid upland cotton (Gossypium hirsutum) is a drought-responsive lincRNA predominantly expressed in the nucleoplasm. Chromatin isolation by RNA purification profiling and electrophoretic mobility shift assay analysis demonstrated that GhDAN1 RNA can bind with DNA fragments containing AAAG motifs, similar to DNA binding with one zinc finger transcription factor binding sequences. The suppression of GhDAN1 mainly regulates genes with AAAG motifs in auxin-response pathways, which are associated with drought stress regulation. As a result, GhDAN1-silenced plants exhibit improved tolerance to drought stress. This phenotype resembles the drought-tolerant phenotype of the A-diploid cotton ancestor species, which has an undetectable expression of DAN1. The role of DAN1 in cotton evolution and drought tolerance regulation suggests that the genomic shock of interspecific hybridization and WGD stimulated neofunctionalization of non-coding genes during the natural evolutionary process.

Efficient chromatin profiling of H3K4me3 modification in cotton using CUT&Tag.

BACKGROUND: In 2019, Kaya-Okur et al. reported on the cleavage under targets and tagmentation (CUT&Tag) technology for efficient profiling of epigenetically modified DNA fragments. It was used mainly for cultured cell lines and was especially effective for small samples and single cells. This strategy generated high-resolution and low-background-noise chromatin profiling data for epigenomic analysis. CUT&Tag is well suited to be used in plant cells, especially in tissues from which small samples are taken, such as ovules, anthers, and fibers. RESULTS: Here, we present a CUT&Tag protocol step by step using plant nuclei. In this protocol, we quantified the nuclei that can be used in each CUT&Tag reaction, and compared the efficiency of CUT&Tag with chromatin immunoprecipitation with sequencing (ChIP-seq) in the leaves of cotton. A general workflow for the bioinformatic analysis of CUT&Tag is also provided. Results indicated that, compared with ChIP-seq, the CUT&Tag procedure was faster and showed a higher-resolution, lower-background signal than did ChIP. CONCLUSION: A CUT&Tag protocol has been refined for plant cells using intact nuclei that have been isolated.

Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe.

BACKGROUND: Gossypium australe F. Mueller (2n = 2x = 26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis. RESULTS: Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds. CONCLUSIONS: The reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.

LncRNAs in polyploid cotton interspecific hybrids are derived from transposon neofunctionalization.

BACKGROUND: Interspecific hybridization and whole genome duplication are driving forces of genomic and organism diversification. But the effect of interspecific hybridization and whole genome duplication on the non-coding portion of the genome in particular remains largely unknown. In this study, we examine the profile of long non-coding RNAs (lncRNAs), comparing them with that of coding genes in allotetraploid cotton (Gossypium hirsutum), its putative diploid ancestors (G. arboreum; G. raimondii), and an F1 hybrid (G. arboreum × G. raimondii, AD). RESULTS: We find that most lncRNAs (80%) that were allelic expressed in the allotetraploid genome. Moreover, the genome shock of hybridization reprograms the non-coding transcriptome in the F1 hybrid. Interestingly, the activated lncRNAs are predominantly transcribed from demethylated TE regions, especially from long interspersed nuclear elements (LINEs). The DNA methylation dynamics in the interspecies hybridization are predominantly associated with the drastic expression variation of lncRNAs. Similar trends of lncRNA bursting are also observed in the progress of polyploidization. Additionally, we find that a representative novel lncRNA XLOC_409583 activated after polyploidization from a LINE in the A subgenome of allotetraploid cotton was involved in control of cotton seedling height. CONCLUSION: Our results reveal that the processes of hybridization and polyploidization enable the neofunctionalization of lncRNA transcripts, acting as important sources of increased plasticity for plants.

Ten alien chromosome additions of Gossypium hirsutum-Gossypium bickii developed by integrative uses of GISH and species-specific SSR markers.

Gossypium bickii: (2n = 26, G1G1), a wild diploid cotton, carries many favourable traits. However, these favourable traits cannot be directly transferred into G. hirsutum (2n = 52, AADD) cultivars due to the differences in genomes. Monosomic alien addition lines (MAALs) are considered an invaluable tool for the introgression of genes of interest from wild relatives into cultivated crops. In this study, the G. hirsutum-G. bickii amphidiploid (2n = 78, AADDG1G1) was backcrossed with G. hirsutum to develop alien additions containing individual G. bickii chromosomes in a G. hirsutum background. Genomic in situ hybridization was employed to detect the number of alien chromosomes added to the backcross progenies. A total of 183 G. bickii-specific DNA markers were developed to discriminate the identities of the G. bickii chromosomes added to G. hirsutum and assess the alien chromosome transmissibility. Chromosomes 4Gb and 13Gb showed the highest transmissibility, while chromosomes 1Gb, 7Gb and 11Gb showed the lowest. Ten of the 13 possible G. hirsutum-G. bickii MAALs were isolated and characterized, which will lay the foundation for transferring resistance genes of G. bickii into G. hirsutum, as well as for gene assignment, physical mapping, and selective isolation and mapping of cDNAs for particular G. bickii chromosomes. The strategies of how to use MAALs to develop varieties with the trait of interest from wild species (such as glanded plant-glandless seed) were proposed and discussed.

Inducement and identification of chromosome introgression and translocation of Gossypium australe on Gossypium hirsutum.

BACKGROUND: We previously reported the development of a set of Gossypium hirsutum-G. australe alien chromosome addition lines. Naturally, however, G. hirsutum-G. australe chromosome exchanges were very limited, impeding the stable transference of useful genes from G. australe (G2G2 genome) into the most cultivated cotton, G. hirsutum (AADD). RESULTS: In the present report, the pollen from a pentaploid (2n = AADDG2) of G. hirsutum-G. australe was irradiated with seven different doses ranging from 10 to 40 Grays and used to pollinate emasculated flowers of G. hirsutum over three consecutive years. Irradiation greatly increased the genetic recombination rates of the G. hirsutum and G. australe chromosomes and a total of 107 chromosome introgression individuals in 192 GISH-negative (with no GISH signal on chromosome) survived individuals, 11 chromosome translocation individuals (containing 12 chromosome translocation events) and 67 chromosome addition individuals were obtained in 70 GISH-positive (with GISH signal(s) on chromosome(s)) survived individuals, which are invaluable for mining desirable genes from G. australe. Multicolor genomic in situ hybridization results showed that there were three types of translocation, whole arm translocation, large alien segment translocation and small alien segment translocation, and that all translocations occurred between the G2-genome and the A-subgenome chromosomes in G. hirsutum. We also found that higher doses induced much higher rates of chromosome variation but also greatly lowered the seed viability and seedling survivability. CONCLUSIONS: Irradiation has been successfully employed to induce chromosome introgressions and chromosome translocations and promote chromosome exchanges between cultivated and wild species. In addition, by balancing the rates of chromosome introgression and translocation to those of seed set, seed germination, and seedling rates in the M1 generation, we conclude that the dosage of 20 Grays is the most suitable. The established methodology may guide the utilization of the tertiary gene pool of Gossypium species such as G. australe in cotton breeding in the future.

Characterization of eleven monosomic alien addition lines added from Gossypium anomalum to Gossypium hirsutum using improved GISH and SSR markers.

BACKGROUND: Gossypium anomalum (BB genome) possesses the desirable characteristics of drought tolerance, resistance to diseases and insect pests, and the potential for high quality fibers. However, it is difficult to transfer the genes associated with these desirable traits into cultivated cotton (G. hirsutum, AADD genome). Monosomic alien addition lines (MAALs) can be used as a bridge to transfer desired genes from wild species into G. hirsutum. In cotton, however, the high number and smaller size of the chromosomes has resulted in difficulties in discriminating chromosomes from wild species in cultivated cotton background, the development of cotton MAALs has lagged far behind many other crops. To date, no set of G. hirsutum-G. anomalum MAALs was reported. Here the amphiploid (AADDBB genome) derived from G. hirsutum × G. anomalum was used to generate a set of G. hirsutum-G. anomalum MAALs through a combination of consecutive backcrossing, genomic in situ hybridization (GISH), morphological survey and microsatellite marker identification. RESULTS: We improved the GISH technique used in our previous research by using a mixture of two probes from G. anomalum and G. herbaceum (AA genome). The results indicate that a ratio of 4:3 (G. anomalum : G. herbaceum) is the most suitable for discrimination of chromosomes from G. anomalum and the At-subgenome of G. hirsutum. Using this improved GISH technique, 108 MAAL individuals were isolated. Next, 170 G. hirsutum- and G. anomalum-specific codominant markers were obtained and employed for characterization of these MAAL individuals. Finally, eleven out of 13 MAALs were identified. Unfortunately, we were unable to isolate Chrs. 1Ba and 5Ba due to their very low incidences in backcrossing generation, as these remained in a condition of multiple additions. CONCLUSIONS: The characterized lines can be employed as bridges for the transfer of desired genes from G. anomalum into G. hirsutum, as well as for gene assignment, isolation of chromosome-specific probes, development of chromosome-specific "paints" for fluorochrome-labeled DNA fragments, physical mapping, and selective isolation and mapping of cDNAs/genes for a particular G. anomalum chromosome.

A New Synthetic Amphiploid (AADDAA) between Gossypium hirsutum and G. arboreum Lays the Foundation for Transferring Resistances to Verticillium and Drought.

Gossypium arboreum, a cultivated cotton species (2n = 26, AA) native to Asia, possesses invaluable characteristics unavailable in the tetraploid cultivated cotton gene pool, such as resistance to pests and diseases and tolerance to abiotic stresses. However, it is quite difficult to transfer favorable traits into Upland cotton through conventional methods due to the cross-incompatibility of G. hirsutum (2n = 52, AADD) and G. arboreum. Here, we improved an embryo rescue technique to overcome the cross-incompatibility between these two parents for transferring favorable genes from G. arboreum into G. hirsutum. Our results indicate that MSB2K supplemented with 0.5 mg l(-1) kinetin and 250 mg(-1) casein hydrolysate is an efficient initial medium for rescuing early (3 d after pollination) hybrid embryos. Eight putative hybrids were successfully obtained, which were further verified and characterized by cytology, molecular markers and morphological analysis. The putative hybrids were subsequently treated with different concentrations of colchicine solution to double their chromosomes. The results demonstrate that four putative hybrid plants were successfully chromosome-doubled by treatment with 0.1% colchicine for 24 h and become amphiploid, which were confirmed by cytological observation, self-fertilization and backcrossing. Preliminary assessments of resistance at seedling stage indicate that the synthetic amphiploid showed highly resistant to Verticillium and drought. The synthetic amphiploid between G. hirsutum × G. arboreum would lay the foundation for developing G. arboreum-introgressed lines with the uniform genetic background of G. hirsutum acc TM-1, which would greatly enhance and simplify the mining, isolation, characterization, cloning and use of G. arboreum-specific desirable genes in future cotton breeding programs.

Identification of centromeric regions on the linkage map of cotton using centromere-related repeats.

Centromere usually contains high-copy-number retrotransposons and satellite repeats, which are difficult to map, clone and sequence. Currently, very little is known about the centromere in cotton. Here, we sequenced a bacterial artificial chromosome (BAC) mapping to the centromeric region and predicted four long-terminal-repeat (LTR) retrotransposons. They were located in the heterochromatic centromeric regions of all 52 pachytene chromosomes in Gossypium hirsutum. Fiber-FISH mapping revealed that these retrotransposons span an area of at least 1.8Mb in the centromeric region. Comparative analysis showed that these retrotransposons generated similar, strong fluorescent signals in the D progenitor Gossypium raimondii but not in the A progenitor Gossypium herbaceum, suggesting that the centromere sequence of tetraploid cotton might be derived from the D progenitor. Centromeric regions were anchored on 13 chromosomes of D-genome sequence. Characterization of these centromere-related repeats and regions will enhance cotton centromere mapping, sequencing and evolutionary studies.